Methods of treating radiation induced gastrointestinal syndrome (RIGS) and related disease states using compound 512

ABSTRACT

The present disclosure is directed to method of treatment for treating or ameliorating various conditions caused by radiation exposure such as RIGS, enteritis, mucositis, e.g., oral mucositis, and proctitis by the administration of compound 512, or an analog thereof.

RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 16/058,943 and claims priority and is entitled to the filingdate of U.S. provisional patent application Ser. No. 62/542,647 filed onAug. 8, 2017. The contents of the aforementioned application areincorporated herein by reference.

BACKGROUND

The risk of large populations encountering radiation exposure is realand growing due to the proliferation of rogue non state actors,political instability resulting in potential access of nuclear weaponsby terrorist, and by natural disaster as evidenced by the release ofradioactive material from the Fukushima nuclear power plant in early2011. Total body exposure to radiation results acute radiation syndromesdescribing a clinical condition with multi organ syndrome. Radiationdoses less than 8 Gy primarily develops hematopoietic injury and can betreated with supportive care with antibiotics, hydration and bone marrowtransplantation. Doses of more than 10 Gy primarily leads togastrointestinal injury resulting diarrhea, dehydration, sepsis andintestinal bleeding with eventual mortality within 10 to 15 dayspost-exposure. High doses of radiation induces the loss of intestinalstem cells (ISC) and thereby impairs epithelial regeneration. Thedamaged intestinal epithelium significantly reduces the mucosalintegrity and promotes systemic influx of bacterial pathogens, resultingsepsis and death. These lethal gastrointestinal symptoms after radiationexposure are collectively known as radiation-induced gastrointestinalsyndrome (RIGS).

There are few FDA approved radio-protectors able to ameliorate RIGS ifapplied prior to radiation exposure. However, no drugs are availablewhich can mitigate RIGS when administered hours or days postirradiation. Considering the logistical barrier and unavoidable delay totreat victims in large casualty settings there is a tremendous need ofsuch therapeutic measures which can be effective even if started daysafter radiation incident.

Dose dependent radiation damage to the intestinal stem cell is theprimary cause of RIGS. It has been reported previously that inhibitionof radiation induced ISC loss mitigates RIGS. This recent studydemonstrated that extracellular vesicle (EV) mediated delivery of WNTrescues ISCs from radiation toxicity and induces intestinal epithelialrepair with the activation of WNT-β catenin signaling. Wnt/β-cateninsignaling plays a major role in ISC self-renewal and proliferation andthereby maintenance of intestinal epithelial homeostasis and repair. WNTligands bind to LRP5/6 and Frizzled co-receptors present on epithelialcrypt cells, leading to β-catenin stabilization and nucleartranslocation where it binds to the nuclear transcription factor TCF4 todrive a gene-expression program that supports stem cell maintenance,proliferation and differentiation. Activation of WNT/β-catenin signalingis also crucial for crypt regeneration following injury. Several reportshave demonstrated that Respondin 1 (RSPO1), an ISC growth factor andLGR5 receptor agonist, activates WNT/βcatenin pathway to repair andregenerate the intestine following chemo-radiation-induced injury. DKK1,a negative regulator WNT/β-catenin pathway, impairs the RSPO1-inducedintestinal regeneration. LGR5 receptor is associated with Frizzled/LrpWnt receptor complex. Genetic deletion of Lgr5 in mouse intestinalinhibits Rspo1 mediated signaling but can be rescued by Wnt pathwayactivation.

The small molecular agent 512 (1-[(4-nitrobenezene)sulfonyl]-4-phenylpiperazine) mitigates RIGS and improves survival when applied afterlethal dose of radiation exposure, preferably within or after 24 hoursof exposure.

Compound 512 is an agonist of Wnt/βcatenin signaling and mitigatesradiation induced intestinal injury by accelerating repair andregeneration of ISCs. Thus, 512 is also useful in mitigatingradiation-induced syndromes related to RIGS and ISC rescue, such asmucositis, e.g., oral mucositis, enteritis, and proctitis. Thesesyndromes are also often an unwanted side-effect of radiation therapyfor cancer and can be treated using compound 512.

SUMMARY

In one embodiment, the invention provides a method of treating radiationinduced gastrointestinal syndrome (RIGS) in a subject in need thereof,the method comprising the step of administering to the subject atherapeutically effective amount of a compound of Formula IIA or ananalog thereof.

In another embodiment, the invention provides a method of treatingradiation induced mucositis in a subject in need thereof, the methodcomprising the step of administering to the subject a therapeuticallyeffective amount of a compound of Formula IIA or an analog thereof.

In another embodiment, the invention provides a method of treatingradiation-induced proctitis in a subject in need thereof, the methodcomprising the step of administering to the subject a therapeuticallyeffective amount of a compound of Formula IIA or an analog thereof.

In another embodiment, the invention provides a method of treatingradiation induced enteritis in a subject in need thereof, the methodcomprising the step of administering to the subject a therapeuticallyeffective amount of a compound of Formula IIA or an analog thereof.

In another embodiment, the invention provides a method of treatingradiation induced hematopoietic syndrome in a subject in need thereof,the method comprising the step of administering to the subject atherapeutically effective amount of a compound of Formula IIA or ananalog thereof.

In one embodiment, the compound is administered to the subject within 48hours of the radiation exposure. In one embodiment, the compound isadministered to the subject after 24 hours of the radiation exposure.

In one embodiment, the analog is selected from the group consisting ofthe compounds of Formula IIB-E. In one embodiment, the compound isFormula IIA.

In one embodiment, the subject received radiation therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings illustrate aspects of the present invention.

FIG. 1 is a view of a histogram of organoids in response to radiationexposure and treatment with 512 after radiation exposure.

FIG. 2 is a view of a histogram demonstrating the higher buddingorganoid to total organoid ratio compared to an irradiated control.

FIG. 3 is a chart showing NCI 60 cancer cell screening with annotationsfor cancer cell lines used in the assay with 512.

FIG. 4 is a chart showing 512 treatment significantly increasesluciferase activity in HEK293 cells compared to vehicle treated cells.

The above described drawing figures illustrate aspects of the inventionin at least one of its exemplary embodiments, which are further definedin detail in the following description. Features, elements, and aspectsof the invention that are referenced by the same numerals in differentfigures represent the same, equivalent, or similar features, elements,or aspects, in accordance with one or more embodiments.

DETAILED DESCRIPTION

Radiation Induced Gastro Intestinal Syndrome (RIGS) limits the survivalof victims in a mass casualty setting from nuclear accidents orterrorism. Currently there is no approved therapy for protecting ormitigating RIGS resulting from direct cytocidal effects on intestinalstem cells. Compound “512,” an anti-neoplastic small molecular agent,activates Wnt-β catenin signaling, a key-signaling pathway forintestinal epithelial homeostasis and regeneration. Therefore, 512 isuseful as an agent for mitigation of RIGS. Furthermore, 512 mitigatesradiation induced mucositis, enteritis and proctitis. It is also usefulfor treating or preventing these radiation syndromes associated withradiation therapy. Analogs of 512 are also useful in the methods of theinvention.

Radiation enteritis is a malfunction of the large and small bowel thatoccurs during or after radiation therapy to the abdomen, pelvis, orrectum. Severity varies, with approximately 15-20% of patients requiringan altered therapeutic course. It is usually self-limiting, oftenresolves within 3 months and frequently only requires supportivemeasures (Do et al. (2011) Gastroenterol Res Practice 2011: 917941).Chronic small bowel radiation disease typically develops between 18months and 6 years after a completed course of radiotherapy but has beenreported to present up to 30 years later (Kountouras and Zavos (2008)World J Gastroenterol 14: 7289-7301). It is a more common entity thanmany doctors think: 90% of patients who receive pelvic radiotherapydevelop a permanent change in their bowel habits (Olopade et al. (2005)Br J Cancer 92: 1663-1670). It is also problematic, 50% of patients withpelvic irradiation describe their quality of life has been adverselyaffected by a variety of GI symptoms with 20-40% (depending on tumortype) rating the effect on quality of life as moderate or severe(Widmark et al., (1994) Cancer 74: 2520-2532; Crook, et al., (1996) URL47: 387-394; Gami, et al. (2003) Aliment Pharmacol Therapeut 18(10), pp.987-994; and Andreyev (2007 Clin Oncol 19: 790-799).

Mucositis, including oral mucositis, is probably the most common,debilitating complication of cancer treatments, particularlychemotherapy and radiation. It can lead to a number of problems,including pain, nutritional problems as a result of inability to eat,and increased risk of infection due to open sores in the mucosa. It hasa significant effect on the patient's quality of life and can bedose-limiting (i.e., requiring a reduction in subsequent chemotherapydoses).

Radiation enteritis is caused by the inflammation of the small and/orlarge intestine from radiation treatments in the belly, sexual organs,or rectum, including treatment for cervical, pancreatic, prostate,uterine, colon and rectal cancer. Proctitis is a related radiationinduced inflammation of the rectum.

The structure of compound 512 is shown below as Formula IIA:

Compound 512 is also known as 1-[(4-nitrobenezene)sulfonyl]-4-phenylpiperazine. Analogs of 512 include compounds of Formula IIB:

wherein:Y¹ and Y² taken together with X form:

and wherein:X is N;G is N;Z is absent or selected from substituted or unsubstituted alkyl,heteroalkyl, alkenyl, or alkynyl;R⁴ is absent or selected from substituted or unsubstituted aryl; andR⁵ and R⁶ are each independently absent or lower alkyl.

In one embodiment, the analog is selected from Formulae IIC-E:

A compound of Formula IIA, or an analog thereof disclosed herein, can beprepared according to established methodology in the art of organicsynthesis. General methods of synthesizing the compound can be found in,e.g., Stuart Warren and Paul Wyatt, Workbook for Organic Synthesis: TheDisconnection Approach, second Edition, Wiley, 2010. Exemplary methodsof making the compound is provided in U.S. Ser. No. 13/813,923 and U.S.Ser. No. 14/889,719, herein incorporated by reference in their entirety.The compound also includes a pharmaceutically acceptable salt thereof, aprodrug thereof, a hydrate thereof, a solvate thereof, or a polymorphiccrystal thereof. The compound may be administered as a pharmaceuticalcomposition.

The present methods may prevent a disease or condition or one or moresymptoms of a disease or condition. As used herein, a therapeutic that“prevents” a disorder or condition refers to a compound that, in astatistical sample, reduces the occurrence of the disorder or conditionin the treated sample relative to an untreated control sample, or delaysthe onset or reduces the severity of one or more symptoms of thedisorder or condition relative to the untreated control sample.

The term “treating” includes prophylactic and/or therapeutic treatments.The term “prophylactic or therapeutic” treatment is art-recognized andincludes administration to the host of one or more of the subjectcompositions. If it is administered prior to clinical manifestation ofthe unwanted condition (e.g., disease or other unwanted state of thehost animal) then the treatment is prophylactic (i.e., it protects thehost against developing the unwanted condition), whereas if it isadministered after manifestation of the unwanted condition, thetreatment is therapeutic (i.e., it is intended to diminish, ameliorate,or stabilize the existing unwanted condition or side effects thereof).

The compositions and methods of the present invention may be utilized totreat an individual in need thereof. In certain embodiments, theindividual is a mammal such as a human, or a non-human mammal. Whenadministered to an animal, such as a human, the composition or thecompound is preferably administered or used as a pharmaceuticalcomposition comprising, for example, a compound of the invention and apharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers are well known in the art andinclude, for example, aqueous solutions such as water or physiologicallybuffered saline or other solvents or vehicles such as glycols, glycerol,oils such as olive oil, or injectable organic esters. In a preferredembodiment, when such pharmaceutical compositions are for humanadministration, particularly for invasive routes of administration(i.e., routes, such as injection or implantation, that circumventtransport or diffusion through an epithelial barrier), the aqueoussolution is pyrogen-free, or substantially pyrogen-free. The excipientscan be chosen, for example, to effect delayed release of an agent or toselectively target one or more cells, tissues or organs. Thepharmaceutical composition can be in dosage unit form such as tablet,capsule (including sprinkle capsule and gelatin capsule), granule,lyophilized for reconstitution, powder, solution, syrup, suppository,injection or the like. The composition can also be present in atransdermal delivery system, e.g., a skin patch.

A pharmaceutical composition disclosed herein may comprise a therapeuticcompound in an amount sufficient to allow customary administration to anindividual. In certain embodiments, a pharmaceutical compositiondisclosed herein may comprise, e.g., at least 5 mg, at least 10 mg, atleast 15 mg, at least 20 mg, at least 25 mg, at least 30 mg, at least 35mg, at least 40 mg, at least 45 mg, at least 50 mg, at least 55 mg, atleast 60 mg, at least 65 mg, at least 70 mg, at least 75 mg, at least 80mg, at least 85 mg, at least 90 mg, at least 95 mg, or at least 100 mgof a therapeutic compound. In certain embodiments, a pharmaceuticalcomposition disclosed herein may comprise, e.g., at least 5 mg, at least10 mg, at least 20 mg, at least 25 mg, at least 50 mg, at least 75 mg,at least 100 mg, at least 200 mg, at least 300 mg, at least 400 mg, atleast 500 mg, at least 600 mg, at least 700 mg, at least 800 mg, atleast 900 mg, at least 1,000 mg, at least 1,100 mg, at least 1,200 mg,at least 1,300 mg, at least 1,400 mg, or at least 1,500 mg of atherapeutic compound. In yet other aspects of this embodiment, apharmaceutical composition disclosed herein may comprise in the rangeof, e.g., about 5 mg to about 100 mg, about 10 mg to about 100 mg, about50 mg to about 150 mg, about 100 mg to about 250 mg, about 150 mg toabout 350 mg, about 250 mg to about 500 mg, about 350 mg to about 600mg, about 500 mg to about 750 mg, about 600 mg to about 900 mg, about750 mg to about 1,000 mg, about 850 mg to about 1,200 mg, or about 1,000mg to about 1,500 mg. In still certain embodiments, a pharmaceuticalcomposition disclosed herein may comprise in the range of, e.g., about10 mg to about 250 mg, about 10 mg to about 500 mg, about 10 mg to about750 mg, about 10 mg to about 1,000 mg, about 10 mg to about 1,500 mg,about 50 mg to about 250 mg, about 50 mg to about 500 mg, about 50 mg toabout 750 mg, about 50 mg to about 1,000 mg, about 50 mg to about 1,500mg, about 100 mg to about 250 mg, about 100 mg to about 500 mg, about100 mg to about 750 mg, about 100 mg to about 1,000 mg, about 100 mg toabout 1,500 mg, about 200 mg to about 500 mg, about 200 mg to about 750mg, about 200 mg to about 1,000 mg, about 200 mg to about 1,500 mg,about 5 mg to about 1,500 mg, about 5 mg to about 1,000 mg, or about 5mg to about 250 mg.

A pharmaceutical composition disclosed herein may comprise a solvent,emulsion or other diluent in an amount sufficient to dissolve atherapeutic compound disclosed herein. In certain embodiments, apharmaceutical composition disclosed herein may comprise a solvent,emulsion or a diluent in an amount of, e.g., less than about 90% (v/v),less than about 80% (v/v), less than about 70% (v/v), less than about65% (v/v), less than about 60% (v/v), less than about 55% (v/v), lessthan about 50% (v/v), less than about 45% (v/v), less than about 40%(v/v), less than about 35% (v/v), less than about 30% (v/v), less thanabout 25% (v/v), less than about 20% (v/v), less than about 15% (v/v),less than about 10% (v/v), less than about 5% (v/v), or less than about1% (v/v). In certain embodiments, a pharmaceutical composition disclosedherein may comprise a solvent, emulsion or other diluent in an amount ina range of, e.g., about 1% (v/v) to 90% (v/v), about 1% (v/v) to 70%(v/v), about 1% (v/v) to 60% (v/v), about 1% (v/v) to 50% (v/v), about1% (v/v) to 40% (v/v), about 1% (v/v) to 30% (v/v), about 1% (v/v) to20% (v/v), about 1% (v/v) to 10% (v/v), about 2% (v/v) to 50% (v/v),about 2% (v/v) to 40% (v/v), about 2% (v/v) to 30% (v/v), about 2% (v/v)to 20% (v/v), about 2% (v/v) to 10% (v/v), about 4% (v/v) to 50% (v/v),about 4% (v/v) to 40% (v/v), about 4% (v/v) to 30% (v/v), about 4% (v/v)to 20% (v/v), about 4% (v/v) to 10% (v/v), about 6% (v/v) to 50% (v/v),about 6% (v/v) to 40% (v/v), about 6% (v/v) to 30% (v/v), about 6% (v/v)to 20% (v/v), about 6% (v/v) to 10% (v/v), about 8% (v/v) to 50% (v/v),about 8% (v/v) to 40% (v/v), about 8% (v/v) to 30% (v/v), about 8% (v/v)to 20% (v/v), about 8% (v/v) to 15% (v/v), or about 8% (v/v) to 12%(v/v).

The final concentration of a therapeutic compound disclosed herein in apharmaceutical composition disclosed herein may be of any suitableconcentration. In certain embodiments, the final concentration of atherapeutic compound in a pharmaceutical composition may be atherapeutically effective amount. In certain embodiments, the finalconcentration of a therapeutic compound in a pharmaceutical compositionmay be, e.g., at least 0.00001 mg/mL, at least 0.0001 mg/mL, at least0.001 mg/mL, at least 0.01 mg/mL, at least 0.1 mg/mL, at least 1 mg/mL,at least 10 mg/mL, at least 25 mg/mL, at least 50 mg/mL, at least 100mg/mL, at least 200 mg/mL, at least 500 mg/mL, at least 700 mg/mL, atleast 1,000 mg/mL, or at least 1,200 mg/mL. In certain embodiments, theconcentration of a therapeutic compound disclosed herein in the solutionmay be, e.g., at most 1,000 mg/mL, at most 1,100 mg/mL, at most 1,200mg/mL, at most 1,300 mg/mL, at most 1,400 mg/mL, at most 1,500 mg/mL, atmost 2,000 mg/mL, at most 2,000 mg/mL, or at most 3,000 mg/mL. Incertain embodiments, the final concentration of a therapeutic compoundin a pharmaceutical composition may be in a range of, e.g., about0.00001 mg/mL to about 3,000 mg/mL, about 0.0001 mg/mL to about 3,000mg/mL, about 0.01 mg/mL to about 3,000 mg/mL, about 0.1 mg/mL to about3,000 mg/mL, about 1 mg/mL to about 3,000 mg/mL, about 250 mg/mL toabout 3,000 mg/mL, about 500 mg/mL to about 3,000 mg/mL, about 750 mg/mLto about 3,000 mg/mL, about 1,000 mg/mL to about 3,000 mg/mL, about 100mg/mL to about 2,000 mg/mL, about 250 mg/mL to about 2,000 mg/mL, about500 mg/mL to about 2,000 mg/mL, about 750 mg/mL to about 2,000 mg/mL,about 1,000 mg/mL to about 2,000 mg/mL, about 100 mg/mL to about 1,500mg/mL, about 250 mg/mL to about 1,500 mg/mL, about 500 mg/mL to about1,500 mg/mL, about 750 mg/mL to about 1,500 mg/mL, about 1,000 mg/mL toabout 1,500 mg/mL, about 100 mg/mL to about 1,200 mg/mL, about 250 mg/mLto about 1,200 mg/mL, about 500 mg/mL to about 1,200 mg/mL, about 750mg/mL to about 1,200 mg/mL, about 1,000 mg/mL to about 1,200 mg/mL,about 100 mg/mL to about 1,000 mg/mL, about 250 mg/mL to about 1,000mg/mL, about 500 mg/mL to about 1,000 mg/mL, about 750 mg/mL to about1,000 mg/mL, about 100 mg/mL to about 750 mg/mL, about 250 mg/mL toabout 750 mg/mL, about 500 mg/mL to about 750 mg/mL, about 100 mg/mL toabout 500 mg/mL, about 250 mg/mL to about 500 mg/mL, about 0.00001 mg/mLto about 0.0001 mg/mL, about 0.00001 mg/mL to about 0.001 mg/mL, about0.00001 mg/mL to about 0.01 mg/mL, about 0.00001 mg/mL to about 0.1mg/mL, about 0.00001 mg/mL to about 1 mg/mL, about 0.001 mg/mL to about0.01 mg/mL, about 0.001 mg/mL to about 0.1 mg/mL, about 0.001 mg/mL toabout 1 mg/mL, about 0.001 mg/mL to about 10 mg/mL, or about 0.001 mg/mLto about 100 mg/mL.

In certain embodiments, a therapeutically effective amount of atherapeutic compound disclosed herein generally is in the range of about0.001 mg/kg/day to about 100 mg/kg/day. In certain embodiments, aneffective amount of a therapeutic compound disclosed herein may be,e.g., at least 0.001 mg/kg/day, at least 0.01 mg/kg/day, at least 0.1mg/kg/day, at least 1.0 mg/kg/day, at least 5.0 mg/kg/day, at least 10mg/kg/day, at least 15 mg/kg/day, at least 20 mg/kg/day, at least 25mg/kg/day, at least 30 mg/kg/day, at least 35 mg/kg/day, at least 40mg/kg/day, at least 45 mg/kg/day, or at least 50 mg/kg/day. In certainembodiments, an effective amount of a therapeutic compound disclosedherein may be in the range of, e.g., about 0.001 mg/kg/day to about 10mg/kg/day, about 0.001 mg/kg/day to about 15 mg/kg/day, about 0.001mg/kg/day to about 20 mg/kg/day, about 0.001 mg/kg/day to about 25mg/kg/day, about 0.001 mg/kg/day to about 30 mg/kg/day, about 0.001mg/kg/day to about 35 mg/kg/day, about 0.001 mg/kg/day to about 40mg/kg/day, about 0.001 mg/kg/day to about 45 mg/kg/day, about 0.001mg/kg/day to about 50 mg/kg/day, about 0.001 mg/kg/day to about 75mg/kg/day, about 0.001 mg/kg/day to about 100 mg/kg/day, about 0.001mg/kg/day to about 150 mg/kg/day, about 0.001 mg/kg/day to about 200mg/kg/day, about 0.001 mg/kg/day to about 250 mg/kg/day, about 0.001mg/kg/day to about 300 mg/kg/day, about 0.001 mg/kg/day to about 350mg/kg/day, about 0.001 mg/kg/day to about 400 mg/kg/day, about 0.001mg/kg/day to about 450 mg/kg/day, about 0.001 mg/kg/day to about 500mg/kg/day, about 0.001 mg/kg/day to about 550 mg/kg/day, about 0.001mg/kg/day to about 600 mg/kg/day, about 0.001 mg/kg/day to about 650mg/kg/day, about 0.001 mg/kg/day to about 700 mg/kg/day, about 0.001mg/kg/day to about 750 mg/kg/day, or about 0.001 mg/kg/day to about 800mg/kg/day. In yet other aspects of this embodiment, an effective amountof a therapeutic compound disclosed herein may be in the range of, e.g.,about 0.01 mg/kg/day to about 10 mg/kg/day, about 0.01 mg/kg/day toabout 15 mg/kg/day, about 0.01 mg/kg/day to about 20 mg/kg/day, about0.01 mg/kg/day to about 25 mg/kg/day, about 0.01 mg/kg/day to about 30mg/kg/day, about 0.01 mg/kg/day to about 35 mg/kg/day, about 0.01mg/kg/day to about 40 mg/kg/day, about 0.01 mg/kg/day to about 45mg/kg/day, about 0.01 mg/kg/day to about 50 mg/kg/day, about 0.01mg/kg/day to about 75 mg/kg/day, about 0.01 mg/kg/day to about 100mg/kg/day, about 0.01 mg/kg/day to about 150 mg/kg/day, about 0.01mg/kg/day to about 200 mg/kg/day, about 0.01 mg/kg/day to about 250mg/kg/day, about 0.01 mg/kg/day to about 300 mg/kg/day, about 0.01mg/kg/day to about 350 mg/kg/day, about 0.01 mg/kg/day to about 400mg/kg/day, about 0.01 mg/kg/day to about 450 mg/kg/day, about 0.01mg/kg/day to about 500 mg/kg/day, about 0.01 mg/kg/day to about 550mg/kg/day, about 0.01 mg/kg/day to about 600 mg/kg/day, about 0.01mg/kg/day to about 650 mg/kg/day, about 0.01 mg/kg/day to about 700mg/kg/day, about 0.01 mg/kg/day to about 750 mg/kg/day, or about 0.01mg/kg/day to about 800 mg/kg/day. In certain embodiments, an effectiveamount of a therapeutic compound disclosed herein may be in the rangeof, e.g., about 0.1 mg/kg/day to about 10 mg/kg/day, about 0.1 mg/kg/dayto about 15 mg/kg/day, about 0.1 mg/kg/day to about 20 mg/kg/day, about0.1 mg/kg/day to about 25 mg/kg/day, about 0.1 mg/kg/day to about 30mg/kg/day, about 0.1 mg/kg/day to about 35 mg/kg/day, about 0.1mg/kg/day to about 40 mg/kg/day, about 0.1 mg/kg/day to about 45mg/kg/day, about 0.1 mg/kg/day to about 50 mg/kg/day, about 0.1mg/kg/day to about 75 mg/kg/day, about 0.1 mg/kg/day to about 100mg/kg/day, about 0.1 mg/kg/day to about 150 mg/kg/day, about 0.1mg/kg/day to about 200 mg/kg/day, about 0.1 mg/kg/day to about 250mg/kg/day, about 0.1 mg/kg/day to about 300 mg/kg/day, about 0.1mg/kg/day to about 350 mg/kg/day, about 0.1 mg/kg/day to about 400mg/kg/day, about 0.1 mg/kg/day to about 450 mg/kg/day, about 0.1mg/kg/day to about 500 mg/kg/day, about 0.1 mg/kg/day to about 550mg/kg/day, about 0.1 mg/kg/day to about 600 mg/kg/day, about 0.1mg/kg/day to about 650 mg/kg/day, about 0.1 mg/kg/day to about 700mg/kg/day, about 0.1 mg/kg/day to about 750 mg/kg/day, or about 0.1mg/kg/day to about 800 mg/kg/day. In certain embodiments, an effectiveamount of a therapeutic compound disclosed herein may be in the rangeof, e.g., about 10 mg/kg/day to about 15 mg/kg/day, about 10 mg/kg/dayto about 20 mg/kg/day, about 10 mg/kg/day to about 25 mg/kg/day, about10 mg/kg/day to about 30 mg/kg/day, about 10 mg/kg/day to about 35mg/kg/day, about 10 mg/kg/day to about 40 mg/kg/day, about 10 mg/kg/dayto about 45 mg/kg/day, about 10 mg/kg/day to about 50 mg/kg/day, about10 mg/kg/day to about 75 mg/kg/day, about 10 mg/kg/day to about 100mg/kg/day, about 10 mg/kg/day to about 150 mg/kg/day, about 10 mg/kg/dayto about 200 mg/kg/day, about 10 mg/kg/day to about 250 mg/kg/day, about10 mg/kg/day to about 300 mg/kg/day, about 10 mg/kg/day to about 350mg/kg/day, about 10 mg/kg/day to about 400 mg/kg/day, about 10 mg/kg/dayto about 450 mg/kg/day, about 10 mg/kg/day to about 500 mg/kg/day, about10 mg/kg/day to about 550 mg/kg/day, about 10 mg/kg/day to about 600mg/kg/day, about 10 mg/kg/day to about 650 mg/kg/day, about 10 mg/kg/dayto about 700 mg/kg/day, about 10 mg/kg/day to about 750 mg/kg/day, orabout 10 mg/kg/day to about 800 mg/kg/day.

In other aspects of this embodiment, an effective amount of atherapeutic compound disclosed herein may be in the range of, e.g.,about 1 mg/kg/day to about 10 mg/kg/day, about 1 mg/kg/day to about 15mg/kg/day, about 1 mg/kg/day to about 20 mg/kg/day, about 1 mg/kg/day toabout 25 mg/kg/day, about 1 mg/kg/day to about 30 mg/kg/day, about 1mg/kg/day to about 35 mg/kg/day, about 1 mg/kg/day to about 40mg/kg/day, about 1 mg/kg/day to about 45 mg/kg/day, about 1 mg/kg/day toabout 50 mg/kg/day, about 1 mg/kg/day to about 75 mg/kg/day, or about 1mg/kg/day to about 100 mg/kg/day. In certain embodiments, an effectiveamount of a therapeutic compound disclosed herein may be in the rangeof, e.g., about 5 mg/kg/day to about 10 mg/kg/day, about 5 mg/kg/day toabout 15 mg/kg/day, about 5 mg/kg/day to about 20 mg/kg/day, about 5mg/kg/day to about 25 mg/kg/day, about 5 mg/kg/day to about 30mg/kg/day, about 5 mg/kg/day to about 35 mg/kg/day, about 5 mg/kg/day toabout 40 mg/kg/day, about 5 mg/kg/day to about 45 mg/kg/day, about 5mg/kg/day to about 50 mg/kg/day, about 5 mg/kg/day to about 75mg/kg/day, or about 5 mg/kg/day to about 100 mg/kg/day.

In liquid and semi-solid formulations, a concentration of a therapeuticcompound disclosed herein typically may be between about 50 mg/mL toabout 1,000 mg/mL. In certain embodiments, a therapeutically effectiveamount of a therapeutic disclosed herein may be from, e.g., about 50mg/mL to about 100 mg/mL, about 50 mg/mL to about 200 mg/mL, about 50mg/mL to about 300 mg/mL, about 50 mg/mL to about 400 mg/mL, about 50mg/mL to about 500 mg/mL, about 50 mg/mL to about 600 mg/mL, about 50mg/mL to about 700 mg/mL, about 50 mg/mL to about 800 mg/mL, about 50mg/mL to about 900 mg/mL, about 50 mg/mL to about 1,000 mg/mL, about 100mg/mL to about 200 mg/mL, about 100 mg/mL to about 300 mg/mL, about 100mg/mL to about 400 mg/mL, about 100 mg/mL to about 500 mg/mL, about 100mg/mL to about 600 mg/mL, about 100 mg/mL to about 700 mg/mL, about 100mg/mL to about 800 mg/mL, about 100 mg/mL to about 900 mg/mL, about 100mg/mL to about 1,000 mg/mL, about 200 mg/mL to about 300 mg/mL, about200 mg/mL to about 400 mg/mL, about 200 mg/mL to about 500 mg/mL, about200 mg/mL to about 600 mg/mL, about 200 mg/mL to about 700 mg/mL, about200 mg/mL to about 800 mg/mL, about 200 mg/mL to about 900 mg/mL, about200 mg/mL to about 1,000 mg/mL, about 300 mg/mL to about 400 mg/mL,about 300 mg/mL to about 500 mg/mL, about 300 mg/mL to about 600 mg/mL,about 300 mg/mL to about 700 mg/mL, about 300 mg/mL to about 800 mg/mL,about 300 mg/mL to about 900 mg/mL, about 300 mg/mL to about 1,000mg/mL, about 400 mg/mL to about 500 mg/mL, about 400 mg/mL to about 600mg/mL, about 400 mg/mL to about 700 mg/mL, about 400 mg/mL to about 800mg/mL, about 400 mg/mL to about 900 mg/mL, about 400 mg/mL to about1,000 mg/mL, about 500 mg/mL to about 600 mg/mL, about 500 mg/mL toabout 700 mg/mL, about 500 mg/mL to about 800 mg/mL, about 500 mg/mL toabout 900 mg/mL, about 500 mg/mL to about 1,000 mg/mL, about 600 mg/mLto about 700 mg/mL, about 600 mg/mL to about 800 mg/mL, about 600 mg/mLto about 900 mg/mL, or about 600 mg/mL to about 1,000 mg/mL.

As used herein, “mitigating” means reducing one or more negativesymptoms of a condition, relative to a cell, organ, tissue, or organismdisplaying the symptom or condition for the same amount of time, butuntreated.

In some embodiments, contacting the cell, organ, tissue, or organism thepresent compounds may comprise administering a therapeutically effectiveamount of the compound to a subject. As used herein, a “therapeuticallyeffective amount” is an amount sufficient to mitigate the negativesymptom or condition.

The subject may be a human, rat, mouse, cat, dog, horse, sheep, cow,monkey, avian, or amphibian. In another embodiment, the cell is in vivoor in vitro. Typical subjects to which compounds of the invention may beadministered will be mammals, particularly primates, especially humans.For veterinary applications, a wide variety of subjects will besuitable, e.g. livestock such as cattle, sheep, goats, cows, swine andthe like; poultry such as chickens, ducks, geese, turkeys, and the like;and domesticated animals particularly pets such as dogs and cats. Fordiagnostic or research applications, a wide variety of mammals will besuitable subjects including rodents (e.g. mice, rats, hamsters),rabbits, primates, and swine such as inbred pigs and the like.Additionally, for in vitro applications, such as in vitro diagnostic andresearch applications, body fluids and cell samples of the abovesubjects will be suitable for use such as mammalian, particularlyprimate such as human, blood, urine or tissue samples, or blood urine ortissue samples of the animals mentioned for veterinary applications.

When administering to an organism, the compound may be administered byany suitable means. In some embodiments, the compounds or formulationsare administered orally. In some embodiments, the compounds orformulations are administered by injection, e.g. subcutaneous,parenteral, or intravenous, injections.

In some embodiments the compound may be administered in combination withother potential mitigators. In a particular embodiment, the compositionmay be administered with growth factors, NSAIDs, chemotherapeutics,anti-inflammatories, antibiotics, Metformin (Glucophage, Glumetza,others), Sulfonylureas, Meglitinides, Thiazolidinediones, DPP-4inhibitors, GLP-1 receptor agonists, SGLT2 inhibitors, and/or Insulintherapy, for the treatment of the above conditions. In one aspect, thegrowth factor can be G-CSF (aka filgrastim, NEUPOGEN®) or erythropoietin(aka EPOGEN®).

In other embodiments, the compositions may comprise an effective amountof a modulator and/or other pharmaceutically active agent in aphysiologically-acceptable carrier. The carrier may take a wide varietyof forms depending on the form of preparation desired for a particularroute of administration. Suitable carriers and their formulation aredescribed, for example, in Remington's Pharmaceutical Sciences by E. W.Martin. In some embodiments, the compound may be contained in anyappropriate amount in any suitable carrier substance, and is generallypresent in an amount of 1-95% by weight of the total weight of thecomposition. The composition may be provided in a dosage form that issuitable for parenteral (e.g., subcutaneously, intravenously,intramuscularly, or intraperitoneally) or oral administration route. Thepharmaceutical compositions may be formulated according to conventionalpharmaceutical practice (see, e.g., Remington: The Science and Practiceof Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams &Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J.Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York).

In some embodiments, the compositions may be in a form suitable foradministration by sterile injection. In one example, to prepare such acomposition, the compositions(s) are dissolved or suspended in aparenterally acceptable liquid vehicle. Among acceptable vehicles andsolvents that may be employed are water, water adjusted to a suitable pHby addition of an appropriate amount of hydrochloric acid, sodiumhydroxide or a suitable buffer, 1,3-butanediol, Ringer's solution, andisotonic sodium chloride solution and dextrose solution. The aqueousformulation may also contain one or more preservatives (e.g., methyl,ethyl or n-propyl p-hydroxybenzoate). For parenteral formulations, thecarrier will usually comprise sterile water, though other ingredients,for example, ingredients that aid solubility or for preservation, may beincluded. Injectable solutions may also be prepared in which caseappropriate stabilizing agents may be employed. In one embodiment, theformulation includes at least one or more of methanesulfonic acid,povidone, benzyl alcohol, n-Methyl pyrrolidone, ethaonol, Poloxamer 188,lactic acid, Captisol (SBE-beta-CD), or Vitamin E, such as TPGS (d-alphatocopheryl polyethylene glycol 1000 succinate).

Formulations suitable for parenteral administration usually comprise asterile aqueous preparation of the compound, which may be isotonic withthe blood of the recipient (e.g., physiological saline solution). Suchformulations may include suspending agents and thickening agents andliposomes or other microparticulate systems which are designed to targetthe compound to blood components or one or more organs. The formulationsmay be presented in unit-dose or multi-dose form.

Parenteral administration may comprise any suitable form of systemicdelivery or localized delivery. Administration may for example beintravenous, intra-arterial, intrathecal, intramuscular, subcutaneous,intramuscular, intra-abdominal (e.g., intraperitoneal), etc., and may beeffected by infusion pumps (external or implantable) or any othersuitable means appropriate to the desired administration modality.

In some embodiments, the compositions may be in a form suitable for oraladministration. In compositions in oral dosage form, any of the usualpharmaceutical media may be employed. Thus, for liquid oralpreparations, such as, for example, suspensions, elixirs and solutions,suitable carriers and additives include water, glycols, oils, alcohols,flavoring agents, preservatives, coloring agents and the like. For solidoral preparations such as, for example, powders, capsules and tablets,suitable carriers and additives include starches, sugars, diluents,granulating agents, lubricants, binders, disintegrating agents and thelike. If desired, tablets may be sugar coated or enteric coated bystandard techniques.

Compositions suitable for oral administration may be presented asdiscrete units such as capsules, cachets, tablets, or lozenges, eachcontaining a predetermined amount of the active ingredient as a powderor granules. Optionally, a suspension in an aqueous liquor or anon-aqueous liquid may be employed, such as a syrup, an elixir, anemulsion, or a draught. Formulations for oral use include tabletscontaining active ingredient(s) in a mixture with pharmaceuticallyacceptable excipients. Such formulations are known to the skilledartisan. Excipients may be, for example, inert diluents or fillers(e.g., sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose,starches including potato starch, calcium carbonate, sodium chloride,lactose, calcium phosphate, calcium sulfate, or sodium phosphate);granulating and disintegrating agents (e.g., cellulose derivativesincluding microcrystalline cellulose, starches including potato starch,croscarmellose sodium, alginates, or alginic acid); binding agents(e.g., sucrose, glucose, sorbitol, acacia, alginic acid, sodiumalginate, gelatin, starch, pregelatinized starch, microcrystallinecellulose, magnesium aluminum silicate, carboxymethylcellulose sodium,methylcellulose, hydroxypropyl methylcellulose, ethylcellulose,polyvinylpyrrolidone, or polyethylene glycol); and lubricating agents,glidants, and antiadhesives (e.g., magnesium stearate, zinc stearate,stearic acid, silicas, hydrogenated vegetable oils, or talc). Otherpharmaceutically acceptable excipients can be colorants, flavoringagents, plasticizers, humectants, buffering agents, and the like.

A syrup may be made by adding the compound to a concentrated aqueoussolution of a sugar, for example sucrose, to which may also be added anyaccessory ingredient(s). Such accessory ingredient(s) may includeflavorings, suitable preservative, agents to retard crystallization ofthe sugar, and agents to increase the solubility of any otheringredient, such as a polyhydroxy alcohol, for example glycerol orsorbitol.

In some embodiments, the composition may be in a form of nasal or othermucosal spray formulations (e.g. inhalable forms). These formulationscan include purified aqueous solutions of the active compounds withpreservative agents and isotonic agents. Such formulations can beadjusted to a pH and isotonic state compatible with the nasal or othermucous membranes. Alternatively, they can be in the form of finelydivided solid powders suspended in a gas carrier. Such formulations maybe delivered by any suitable means or method, e.g., by nebulizer,atomizer, metered dose inhaler, or the like.

In some embodiments, the composition may be in a form suitable forrectal administration. These formulations may be presented as asuppository with a suitable carrier such as cocoa butter, hydrogenatedfats, or hydrogenated fatty carboxylic acids.

In some embodiments, the composition may be in a form suitable fortransdermal administration. These formulations may be prepared, forexample, by incorporating the active compound in a thixotropic orgelatinous carrier such as a cellulosic medium, e.g., methyl celluloseor hydroxyethyl cellulose, with the resulting formulation then beingpacked in a transdermal device adapted to be secured in dermal contactwith the skin of a wearer.

In addition to the aforementioned ingredients, compositions of theinvention may further include one or more accessory ingredient(s)selected from encapsulants, diluents, buffers, flavoring agents,binders, disintegrants, surface active agents, thickeners, lubricants,preservatives (including antioxidants), and the like.

In some embodiments, compositions may be formulated for immediaterelease, sustained release, delayed-onset release or any other releaseprofile known to one skilled in the art. In some embodiments, thepharmaceutical composition may be formulated to release the activecompound substantially immediately upon administration or at anypredetermined time or time period after administration. The latter typesof compositions are generally known as controlled release formulations,which include (i) formulations that create a substantially constantconcentration of the drug within the body over an extended period oftime; (ii) formulations that after a predetermined lag time create asubstantially constant concentration of the drug within the body over anextended period of time; (iii) formulations that sustain action during apredetermined time period by maintaining a relatively constant,effective level in the body with concomitant minimization of undesirableside effects associated with fluctuations in the plasma level of theactive substance (sawtooth kinetic pattern); (iv) formulations thatlocalize action by, e.g., spatial placement of a controlled releasecomposition adjacent to or in the central nervous system orcerebrospinal fluid; (v) formulations that allow for convenient dosing,such that doses are administered, for example, once every one or twoweeks; and (vi) formulations that target the site of a pathology. Forsome applications, controlled release formulations obviate the need forfrequent dosing to sustain activity at a medically advantageous level.

Any of a number of strategies can be pursued in order to obtaincontrolled release in which the rate of release outweighs the rate ofmetabolism of the compound in question. In one example, controlledrelease is obtained by appropriate selection of various formulationparameters and ingredients, including, e.g., various types of controlledrelease compositions and coatings. Thus, the compound is formulated withappropriate excipients into a pharmaceutical composition that, uponadministration, releases the compound in a controlled manner. Examplesinclude single or multiple unit tablet or capsule compositions, oilsolutions, suspensions, emulsions, microcapsules, microspheres,molecular complexes, nanoparticles, patches, and liposomes.

In some embodiments, the composition may comprise a “vectorized” form,such as by encapsulation of the compound in a liposome or otherencapsulate medium, or by fixation of the compound, e.g., by covalentbonding, chelation, or associative coordination, on a suitablebiomolecule, such as those selected from proteins, lipoproteins,glycoproteins, and polysaccharides.

In some embodiments, the composition can be incorporated intomicrospheres, microcapsules, nanoparticles, liposomes, or the like forcontrolled release. Furthermore, the composition may include suspending,solubilizing, stabilizing, pH-adjusting agents, tonicity adjustingagents, and/or dispersing, agents. Alternatively, the compound may beincorporated in biocompatible carriers, implants, or infusion devices.

Materials for use in the preparation of microspheres and/ormicrocapsules are, e.g., biodegradable/bioerodible polymers such aspolygalactin, poly-(isobutyl cyanoacrylate),poly(2-hydroxyethyl-L-glutamine) and, poly(lactic acid). Biocompatiblecarriers that may be used when formulating a controlled releaseparenteral formulation are carbohydrates (e.g., dextrans), proteins(e.g., albumin), lipoproteins, or antibodies. Materials for use inimplants can be non-biodegradable (e.g., polydimethyl siloxane) orbiodegradable (e.g., poly(caprolactone), poly(lactic acid),poly(glycolic acid) or poly(ortho esters) or combinations thereof).

In all embodiments, the compound or other active compounds may bepresent as pharmaceutically acceptable salts or other derivatives, suchas ether derivatives, ester derivatives, acid derivatives, and aqueoussolubility altering derivatives of the active compound. Derivativesinclude all individual enantiomers, diastereomers, racemates, and otherisomers of the compounds. Derivatives also include all polymorphs andsolvates, such as hydrates and those formed with organic solvents, ofthe compounds. Such isomers, polymorphs, and solvates may be prepared bymethods known in the art, such as by regiospecific and/orenantioselective synthesis and resolution.

The ability to prepare salts depends on the acidity or basicity of thecompounds. Suitable salts of the compounds include, but are not limitedto, acid addition salts, such as those made with hydrochloric,hydrobromic, hydroiodic, perchloric, sulfuric, nitric, phosphoric,acetic, propionic, glycolic, lactic pyruvic, malonic, succinic, maleic,fumaric, malic, tartaric, citric, benzoic, carbonic, cinnamic, mandelic,methanesulfonic, ethanesulfonic, hydroxyethanesulfonic,benezenesulfonic, p-toluene sulfonic, cyclohexanesulfamic, salicyclic,p-aminosalicylic, 2-phenoxybenzoic, and 2-acetoxybenzoic acid; saltsmade with saccharin; alkali metal salts, such as sodium and potassiumsalts; alkaline earth metal salts, such as calcium and magnesium salts;and salts formed with organic or inorganic ligands, such as quaternaryammonium salts.

Additional suitable salts include, but are not limited to, acetate,benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate,bromide, calcium edetate, camsylate, carbonate, chloride, clavulanate,citrate, dihydrochloride, edetate, edisylate, estolate, esylate,fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate,hexylresorcinate, hydrabamine, hydrobromide, hydrochloride,hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate,malate, maleate, mandelate, mesylate, methylbromide, methylnitrate,methylsulfate, mucate, napsylate, nitrate, N-methylglucamine ammoniumsalt, oleate, pamoate (embonate), palmitate, pantothenate,phosphate/diphosphate, polygalacturonate, salicylate, stearate, sulfate,subacetate, succinate, tannate, tartrate, teoclate, tosylate,triethiodide and valerate salts of the compounds.

The pharmaceutically acceptable acid addition salts can also exist asvarious solvates, such as with water, methanol, ethanol,dimethylformamide, and the like. Mixtures of such solvates can also beprepared. The source of such solvate can be from the solvent ofcrystallization, inherent in the solvent of preparation orcrystallization, or adventitious to such solvent.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: (1)water-soluble antioxidants, such as ascorbic acid, cysteinehydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfiteand the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate,butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),lecithin, propyl gallate, alpha-tocopherol, and the like; and (3)metal-chelating agents, such as citric acid, ethylenediamine tetraaceticacid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Unless the context clearly indicates otherwise, compositions of allembodiments can comprise various pharmaceutically acceptable salts, orother derivatives described above.

The formulation and preparation of such compositions are well known tothose skilled in the art of pharmaceutical formulation. Formulations canbe found in Remington: The Science and Practice of Pharmacy.

The amount of the compound employed in the present invention to be usedvaries according to the condition, the patient/subject, and the extentof the condition.

The contents of all cited references (including literature references,issued patents, published patent applications) as cited throughout thisapplication are hereby expressly incorporated by reference. Theinvention and the manner and process of making and using it, aredescribed in such full, clear, concise and exact terms as to enable anyperson skilled in the art to which it pertains, to make and use thesame.

The term “unit dosage form” or “unit” as used herein refers tophysically discrete units suitable as unitary dosages for human andanimal subjects, each unit containing a predetermined quantity of thecompound calculated in an amount sufficient to produce the desiredeffect in association with a pharmaceutically acceptable, diluent,carrier or vehicle. The specifications for the novel unit dosage formsof the present invention depend on the particular compound employed andthe effect to be achieved, and the pharmacodynamics associated with eachcompound in the subject.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions may be varied so as to obtain an amount of the activeingredient that is effective to achieve the desired therapeutic responsefor a particular patient, composition, and mode of administration,without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound or combination ofcompounds employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound(s) being employed, the duration of the treatment,other drugs, compounds and/or materials used in combination with theparticular compound(s) employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the therapeutically effective amount of thepharmaceutical composition required. For example, the physician orveterinarian could start doses of the pharmaceutical composition orcompound at levels lower than that required in order to achieve thedesired therapeutic effect and gradually increase the dosage until thedesired effect is achieved. By “therapeutically effective amount” ismeant the concentration of a compound that is sufficient to elicit thedesired therapeutic effect. It is generally understood that theeffective amount of the compound will vary according to the weight, sex,age, and medical history of the subject. Other factors which influencethe effective amount may include, but are not limited to, the severityof the patient's condition, the disorder being treated, the stability ofthe compound, and, if desired, another type of therapeutic agent beingadministered with the compound of the invention. A larger total dose canbe delivered by multiple administrations of the agent. Methods todetermine efficacy and dosage are known to those skilled in the art(Isselbacher et al. (1996) Harrison's Principles of Internal Medicine 13ed., 1814-1882, herein incorporated by reference).

In general, a suitable daily dose of an active compound used in thecompositions and methods of the invention will be that amount of thecompound that is the lowest dose effective to produce a therapeuticeffect. Such an effective dose will generally depend upon the factorsdescribed above.

Dosing can be single dosage or cumulative (serial dosing), and can bereadily determined by one skilled in the art. For instance, treatmentmay comprise a one-time administration of an effective dose of apharmaceutical composition disclosed herein. Alternatively, treatmentmay comprise multiple administrations of an effective dose of apharmaceutical composition carried out over a range of time periods,such as, e.g., once daily, twice daily, thrice daily, once every fewdays, or once weekly. The timing of administration can vary fromindividual to individual, depending upon such factors as the severity ofan individual's symptoms. For example, an effective dose of apharmaceutical composition disclosed herein can be administered to anindividual once daily for an indefinite period of time, or until theindividual no longer requires therapy. A person of ordinary skill in theart will recognize that the condition of the individual can be monitoredthroughout the course of treatment and that the effective amount of apharmaceutical composition disclosed herein that is administered can beadjusted accordingly.

If desired, the effective daily dose of the active compound may beadministered as one, two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms. In certain embodiments of the presentinvention, the active compound may be administered two or three timesdaily. In preferred embodiments, the active compound will beadministered once daily.

In certain embodiments, the period of administration of a therapeuticcompound is for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks,12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10months, 11 months, 12 months, or more. In certain embodiments, atreatment regimen may comprise a period during which administration isstopped for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks,12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10months, 11 months, 12 months, or more.

The patient receiving this treatment is any animal in need, includingprimates, in particular humans, and other mammals such as equines,cattle, swine and sheep; and poultry and pets in general.

In other embodiments, the compounds described herein may be providedwith the one or more additional therapeutic agents in a kit, e.g., asseparate pharmaceutical formulations capable of being used together in aconjoint therapy as discussed herein, either together in a singlecontainer or in separate containers. In certain such embodiments, thekit may further include instructions for the conjoint administration ofthe pharmaceutical formulations, e.g., for treating or preventing any ofthe conditions discussed above.

Such combination products may employ compounds of this invention, orpharmaceutically acceptable salts thereof, within the dosage rangedescribed hereinbefore and the other pharmaceutically-active agentwithin its approved dosage range.

In some embodiments, the compound may be administered after thepredicate event, such as after exposure to ionizing radiation, or afterthe initiation of exposure to radiation including accidental ortherapeutic radiation. In one embodiment, the compound is administeredimmediately after the exposure. In another embodiment, the compound isadministered within 12 hours of the exposure. In another embodiment, thecompound is administered within 24 hours of the exposure. In anotherembodiment, the compound is administered at 24 hours after the exposure.In another embodiment, the compound is administered after 24 hours ofexposure. In another embodiment, the compound is administered after 36hours of exposure. In another embodiment, the compound is administeredwithin 48 hours of exposure. In another embodiment, the compound isadministered within 60 hours of exposure. In another embodiment, thecompound is administered within 72 hours of the exposure. In anotherembodiment, the compound is administered within 84 hours of theexposure.

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention and are not intended to limit the invention.

Examples

The following non-limiting examples are provided for illustrativepurposes only in order to facilitate a more complete understanding ofrepresentative embodiments now contemplated. These examples should notbe construed to limit any of the embodiments described in the presentspecification, including those pertaining to the compounds,pharmaceutical compositions, or methods and uses disclosed herein.

Compound 512 Will Mitigate RIGS and Improve Survival Following LethalDose of Radiation

Lethality from Acute Radiation Syndrome (ARS) depends upon dosedependent injury to various organs. Total body exposure to a radiationdose higher than 8 Gy results in mortality within 15 days post exposure,primarily due to RIGS. Intestinal epithelium is highly radio-sensitivebecause of its rapid self-renewal rate compared to any other organ.Every 4-5 days a new epithelium takes charge for mucosal defense undervery strict epithelial homeostasis. High doses of radiation disrupt thishomeostatic balance, kill ISCs and impair the repair process, resultingcomplete loss of mucosal barrier within 5-10 days post-exposure.

512 Mitigates Radiation Injury in Human Lung Organoids

Human non-malignant pulmonary surgical specimens were collected fromKUMC biorepository and then used to develop lung organoids. Human lungorganoids receiving irradiation demonstrated significant loss in buddingstructure. However, treatment with 512 rescued those organoids fromradiation toxicity improves the budding structure.

FIGS. 1 and 2 illustrate that 512 mitigates radiation toxicity in humanpulmonary organoids. Lung organoids were developed from humannon-malignant surgical specimen of lung. Organoids were grown in ALIculture. FIG. 1 : Note loss of budding structure in these organoids inresponse to radiation exposure. However, treatment with 512 afterradiation exposure rescued these organoids from radiation toxicity andimproves budding structure. FIG. 2 : Histogram demonstrating the higherbudding organoid to total organoid ratio compared to irradiated control(p<0.004) (unpaired two tailed t test).

To examine the radio-mitigating role of 512 against RIGS, C57BI6 miceare exposed to graded doses of abdominal irradiation (AIR) (14-15 Gy)after shielding the thorax, head and neck, and extremities, thusprotecting the bone marrow. A single fraction of 14-15 Gy AIR inducesRIGS and lethality in 100% of animals within 7-12 days post-exposure.Mice will receive 512 at 24 hrs post AIR and are expected to continue tosurvive beyond 30 days post-exposure without showing any symptoms ofRIGS. These results will indicate that 512 mitigates the lethalradiation injury in intestine.

Unless there has been a very focal radiation exposure there will also beinvolvement of many other organ systems, and their differentialresponses to various doses of irradiation will impact thegastrointestinal acute radiation syndrome (GI-ARS) dose response. Allradiation doses inducing GI-ARS will have a major impact on the bonemarrow, which in turn will affect the levels of intestinal inflammationand ability of the body to manage the infection resulting from bacterialtranslocation through an impaired intestinal mucosal barrier.

TABLE 1 Cytokine Ctl LTI LTI + 512 IL-6 7 15 88 KC 10 210 245 MCP-1 17103 77 IL-1b 32 50 7 MIP-1b 25 88 13 IL-23 13 79 23

Table 1 shows plasma cytokine levels in rodents treated with 5 mg/kg 512and plasma cytokines tested. Ctl, control with no treatment. LTI, lowerthoracic radiation, LTI+512, radiation, and 512. The cytokine phenotypeis anti-inflammatory where IL-6 is an anti-inflammatory cytokinerequired for controlling local or systemic acute inflammatory responsesand IL-1 is pro inflammatory and so forth.

To investigate involvement of bone marrow in survival outcome upon 512treatment, C57BI6 mice will be exposed to partial body irradiation (PBI)where 40% of total bone marrow is exposed (BM40) to irradiation aftershielding head and fore limbs. Treatment with 512 at 24 hrs postexposure of 14.5 Gy PBI will rescue mice from radiation lethality. Thisdata will indicate that 512 will rescue GI epithelium from GI epitheliumeven in absence of protective function from bone marrow (BM).

Compound 512 Will not Protect Malignant Tissue from Radiation

Compound 512 was examined in the National Cancer Institute (NCI) 60cancer cell line platform. Several of these cell lines are known to bepositive for dysregulation of the Wnt/b-catenin signaling pathway. Noneof these cancer cells will show any proliferative response to 512treatment unlike normal tissue.

NCI 60 cancer cell screening with annotations for cancer cell lines usedin the assay. Each data point represents the percent growth over controlwhich is only vehicle treated (DMSO) as presented with FIG. 3 . The dataclearly indicate that 512 does not promote tumor growth.

These results indicate that 512 can also be used as adjuvant therapy tominimize the toxic side effects of abdominal radiotherapy.

512 Activates Canonical WNT-β Catenin Signaling

To determine the canonical WNT activity in 512 (28.78 μM) HEK293 cellshaving TCF/LEF luciferase reporter construct were treated with 512 orvehicle control. LiCl (10 mM) treatment was used as positive control forluciferase activity. Luciferase activity was determined 24 hr afterusing Dual-Luciferase® Reporter Assay System (Promega) as permanufacturer's protocol. HEK293 cells having FOPFLASH construct wereused as negative control. 512 treatment as presented with FIG. 4 ,significantly increases luciferase activity in HEK293 cells compared tovehicle treated cells. LiCl used as positive control also demonstratedsignificant increase in Luciferase activity. WNT-β catenin signaling iswell known to promote the self renewal and differentiation of LGR5+epithelial (lung, oral, intestinal) stem cells.

In closing, it is to be understood that although aspects of the presentspecification are highlighted by referring to specific embodiments, oneskilled in the art will readily appreciate that these disclosedembodiments are only illustrative of the principles of the subjectmatter disclosed herein. Therefore, it should be understood that thedisclosed subject matter is in no way limited to a particular compound,composition, article, apparatus, methodology, protocol, and/or reagent,etc., described herein, unless expressly stated as such. In addition,those of ordinary skill in the art will recognize that certain changes,modifications, permutations, alterations, additions, subtractions andsub-combinations thereof can be made in accordance with the teachingsherein without departing from the spirit of the present specification.It is therefore intended that the following appended claims and claimshereafter introduced are interpreted to include all such changes,modifications, permutations, alterations, additions, subtractions andsub-combinations as are within their true spirit and scope.

Certain embodiments of the present invention are described herein,including the best mode known to the inventors for carrying out theinvention. Of course, variations on these described embodiments willbecome apparent to those of ordinary skill in the art upon reading theforegoing description. The inventor expects skilled artisans to employsuch variations as appropriate, and the inventors intend for the presentinvention to be practiced otherwise than specifically described herein.Accordingly, this invention includes all modifications and equivalentsof the subject matter recited in the claims appended hereto as permittedby applicable law. Moreover, any combination of the above-describedembodiments in all possible variations thereof is encompassed by theinvention unless otherwise indicated herein or otherwise clearlycontradicted by context.

Groupings of alternative embodiments, elements, or steps of the presentinvention are not to be construed as limitations. Each group member maybe referred to and claimed individually or in any combination with othergroup members disclosed herein. It is anticipated that one or moremembers of a group may be included in, or deleted from, a group forreasons of convenience and/or patentability. When any such inclusion ordeletion occurs, the specification is deemed to contain the group asmodified thus fulfilling the written description of all Markush groupsused in the appended claims.

Unless otherwise indicated, all numbers expressing a characteristic,item, quantity, parameter, property, term, and so forth used in thepresent specification and claims are to be understood as being modifiedin all instances by the term “about.” As used herein, the term “about”means that the characteristic, item, quantity, parameter, property, orterm so qualified encompasses a range of plus or minus ten percent aboveand below the value of the stated characteristic, item, quantity,parameter, property, or term. Accordingly, unless indicated to thecontrary, the numerical parameters set forth in the specification andattached claims are approximations that may vary. For instance, as massspectrometry instruments can vary slightly in determining the mass of agiven analyte, the term “about” in the context of the mass of an ion orthe mass/charge ratio of an ion refers to +/−0.50 atomic mass unit. Atthe very least, and not as an attempt to limit the application of thedoctrine of equivalents to the scope of the claims, each numericalindication should at least be construed in light of the number ofreported significant digits and by applying ordinary roundingtechniques.

Use of the terms “may” or “can” in reference to an embodiment or aspectof an embodiment also carries with it the alternative meaning of “maynot” or “cannot.” As such, if the present specification discloses thatan embodiment or an aspect of an embodiment may be or can be included aspart of the inventive subject matter, then the negative limitation orexclusionary proviso is also explicitly meant, meaning that anembodiment or an aspect of an embodiment may not be or cannot beincluded as part of the inventive subject matter. In a similar manner,use of the term “optionally” in reference to an embodiment or aspect ofan embodiment means that such embodiment or aspect of the embodiment maybe included as part of the inventive subject matter or may not beincluded as part of the inventive subject matter. Whether such anegative limitation or exclusionary proviso applies will be based onwhether the negative limitation or exclusionary proviso is recited inthe claimed subject matter.

Notwithstanding that the numerical ranges and values setting forth thebroad scope of the invention are approximations, the numerical rangesand values set forth in the specific examples are reported as preciselyas possible. Any numerical range or value, however, inherently containscertain errors necessarily resulting from the standard deviation foundin their respective testing measurements. Recitation of numerical rangesof values herein is merely intended to serve as a shorthand method ofreferring individually to each separate numerical value falling withinthe range. Unless otherwise indicated herein, each individual value of anumerical range is incorporated into the present specification as if itwere individually recited herein.

The terms “a,” “an,” “the” and similar references used in the context ofdescribing the present invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. Further, ordinal indicators—such as “first,” “second,” “third,”etc.—for identified elements are used to distinguish between theelements, and do not indicate or imply a required or limited number ofsuch elements, and do not indicate a particular position or order ofsuch elements unless otherwise specifically stated. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein is intended merely to better illuminate the presentinvention and does not pose a limitation on the scope of the inventionotherwise claimed. No language in the present specification should beconstrued as indicating any non-claimed element essential to thepractice of the invention.

When used in the claims, whether as filed or added per amendment, theopen-ended transitional term “comprising” (and equivalent open-endedtransitional phrases thereof like including, containing and having)encompasses all the expressly recited elements, limitations, stepsand/or features alone or in combination with unrecited subject matter;the named elements, limitations and/or features are essential, but otherunnamed elements, limitations and/or features may be added and stillform a construct within the scope of the claim. Specific embodimentsdisclosed herein may be further limited in the claims using theclosed-ended transitional phrases “consisting of” or “consistingessentially of” in lieu of or as an amended for “comprising.” When usedin the claims, whether as filed or added per amendment, the closed-endedtransitional phrase “consisting of” excludes any element, limitation,step, or feature not expressly recited in the claims. The closed-endedtransitional phrase “consisting essentially of” limits the scope of aclaim to the expressly recited elements, limitations, steps and/orfeatures and any other elements, limitations, steps and/or features thatdo not materially affect the basic and novel characteristic(s) of theclaimed subject matter. Thus, the meaning of the open-ended transitionalphrase “comprising” is being defined as encompassing all thespecifically recited elements, limitations, steps and/or features aswell as any optional, additional unspecified ones. The meaning of theclosed-ended transitional phrase “consisting of” is being defined asonly including those elements, limitations, steps and/or featuresspecifically recited in the claim whereas the meaning of theclosed-ended transitional phrase “consisting essentially of” is beingdefined as only including those elements, limitations, steps and/orfeatures specifically recited in the claim and those elements,limitations, steps and/or features that do not materially affect thebasic and novel characteristic(s) of the claimed subject matter.Therefore, the open-ended transitional phrase “comprising” (andequivalent open-ended transitional phrases thereof) includes within itsmeaning, as a limiting case, claimed subject matter specified by theclosed-ended transitional phrases “consisting of” or “consistingessentially of.” As such embodiments described herein or so claimed withthe phrase “comprising” are expressly or inherently unambiguouslydescribed, enabled and supported herein for the phrases “consistingessentially of” and “consisting of.”

All patents, patent publications, and other publications referenced andidentified in the present specification are individually and expresslyincorporated herein by reference in their entirety for the purpose ofdescribing and disclosing, for example, the compositions andmethodologies described in such publications that might be used inconnection with the present invention. These publications are providedsolely for their disclosure prior to the filing date of the presentapplication. Nothing in this regard should be construed as an admissionthat the inventors are not entitled to antedate such disclosure byvirtue of prior invention or for any other reason. All statements as tothe date or representation as to the contents of these documents isbased on the information available to the applicants and does notconstitute any admission as to the correctness of the dates or contentsof these documents.

Lastly, the terminology used herein is for the purpose of describingparticular embodiments only and is not intended to limit the scope ofthe present invention, which is defined solely by the claims.Accordingly, the present invention is not limited to that precisely asshown and described.

What is claimed is:
 1. A method of treating chemotherapy induced injuryor radiation induced injury of the GI tract in a subject in needthereof, the method comprising the step of administering to the subjecta therapeutically effective amount of a compound of Formula IIA:


2. The method of claim 1, wherein the compound is administered to thesubject within 48 hours of the radiation exposure.
 3. The method ofclaim 1, wherein the compound is administered to the subject after 24hours of the radiation exposure.
 4. The method of claim 1, wherein thesubject received radiation therapy or chemotherapy.
 5. The method ofclaim 1, wherein the injury of the GI tract is identified as one or moreof radiation-induced gastrointestinal syndrome (RIGS), radiation-inducedmucositis, radiation-induced proctitis or radiation-induced enteritis.6. The method of claim 1, wherein the injury of the GI tract isidentified as one or more of chemotherapy-induced gastrointestinalsyndrome, chemotherapy-induced mucositis, chemotherapy-induced proctitisor chemotherapy-induced enteritis.
 7. A method of treating chemotherapyinduced injury or radiation induced injury of the GI tract in a subjectin need thereof, the method comprising the step of administering to thesubject a therapeutically effective amount of an analog of Formula IIA,wherein the analog is selected from the group consisting of Formula IIC,Formula IID, Formula IIE and Formula IIB:

wherein Y¹ and Y² taken together with X form:

wherein X is N, G is N, Z is absent or selected from substituted orunsubstituted alkyl, heteroalkyl, alkenyl, or alkynyl, wherein R⁴ isabsent or selected from substituted or unsubstituted aryl; and whereinR⁵ and R⁶ are each independently absent or lower alkyl.
 8. The method ofclaim 7, wherein the compound is administered to the subject within 48hours of the radiation exposure.
 9. The method of claim 7, wherein thecompound is administered to the subject after 24 hours of the radiationexposure.
 10. The method of claim 7, wherein the subject receivedradiation therapy or chemotherapy.
 11. The method of claim 7, whereinthe injury of the GI tract is identified as one or more ofradiation-induced gastrointestinal syndrome (RIGS), radiation-inducedmucositis, radiation-induced proctitis or radiation-induced enteritis.12. The method of claim 7, wherein the injury of the GI tract isidentified as one or more of chemotherapy-induced gastrointestinalsyndrome, chemotherapy-induced mucositis, chemotherapy-induced proctitisor chemotherapy-induced enteritis.